This invention relates to a process for cultivating a microorganism which comprises cultivating a microorganism transformed with a recombinant plasmid containing the desired structural gene derived from an animal, plant or microorganism which can be expressed by a promoter in a tryptophanase operon, and thereby producing large amounts of the desired gene product, to novel recombinant plasmids which can be used in the process, and to microorganism transformed with the plasmids.
There have recently been striking advances in the DNA recombinant technology of producing large amounts of useful substances by using host microorganisms containing recombinant plasmids which include homologous or heterologous DNA fragments bearing encoding the genetic information of useful substances derived from animals, plants or microorganisms. Production of insulin or interferon using Escherichia coli as a host microorganism has already been under way.
In spite of the advances in the DNA recombinant technology, however, no process for producing large amounts of the desired useful substance using recombinant plasmids containing the desired structural gene has yet been established, and the development of a process for cultivating transformants efficiently is urgently in need.
On the other hand, the regulation of expression of a tryptophanase operon (TNa) is controlled by a mechanism, called "catabolite repression" (J. L. Botsford and R. D. DeMoss; J. Bacteriol, 1971, 105, 303-312). It is known that in a culture medium containing glucose as a main carbon source, the expression of the tryptophanase structural gene is very strongly incubated.
Although the promoter in the tryptophanase operon has a high potential to express the desired structural gene, it is necessary to use an expensive carbon source for cultivation such as succinic acid instead of glucose. It is a great problem to develop an efficient gene expression system potentiated by the tna promoter that uses glucose, which is cheap and industrially valuable, but up to the present invention it could not be used as a carbon source for cultivation.
The present inventors extensively studied the effective utilization of the promoter in the tryptophanase operon (tna), and consequently found that by placing a DNA fragment (b) containing the structural gene downstream of a DNA fragment (a) containing both a tna promoter and a regulatory gene tnaC in this order, not only the tryptophanase structural gene but also a foreign structural gene can be expressed strongly by the above promoter.
The regulatory gene tnaC is a short coding region which is located between the tryptophanase promoter and tryptophanase structural gene tnaA and is responsible for the regulation of expression of the tryptophanase operon through a mode, called "transcriptional antitermination" [V. Stewart and C. Yanofsky; J. Bacteriol., 164 (1985) 731-740].
In spite of the fact that a microorganism transformed with a recombinant plasmid containing at least the DNA fragments (a) and (b) undergoes the above-mentioned catabolite repression (the inhibition of gene expression by glucose), if glucose is added continuously or intermittently so that its concentration is maintained at a specified low value in a culture medium containing L- or DL-tryptophan, the present inventors surprisingly found that the transformed microorganism does not undergo catabolite repression, and the desired gene was expressed highly, and the expression product can be obtained in large amounts.
As a method of cultivating a microorganism while glucose is added to the culture medium continuously or intermittently in some low concentrations, Japanese Patent Publication No. 42555/1986 already disclosed a method of cultivating a tryptophan synthase-producing Escherichia coli strain by adding glucose continuously or intermittently so that its concentration in the culture medium is maintained at 1% or less.
It is known that the expression of a tryptophan synthase gene is controlled by a promoter in a tryptophan operon, and this gene expression mechanism does not undergo catabolite repression.
In fact, Agr. Biol. Chem., 38 (7), 1335-1334, 1974 states that when tryptophan synthase-producing Escherichia coli, was cultivated in a medium containing glucose at 0.5 and 1.8% concentration, the tryptophan synthase activity of the cells was 2.4 and 7.3 U/ml respectively, showing that the enzyme activity increases as the concentration of glucose increases. Japanese Patent Application Publication No. 268175/1986 discloses that when cultivating the tryptophan synthase-producing Escherichia coli with a concentration of glucose in the culture medium maintained at 0.5 to 5%, the tryptophan synthase activity shows the highest value.
The above-cited Japanese Patent Publication No. 42555/1986 discloses in the example, that, compared to the values attained at the glucose concentration in the medium initially set at 5.0%, cells yield increased to 2.69 times and the enzyme activity increased 1.80 times (glucose concentration of 1%), cell yield and enzyme activity increased 3.1 times and 2.15 times for a (glucose concentration maintained 0.05%). From the results, at lower concentrations of glucose, both the cells yield and the enzyme activity tended to show an increase. But the difference is only slight. There is no disclosure that tryptophan synthase gene is expressed by the promoter of the tryptophanase operon (tna). Furthermore, when the gene expression is inhibited by catabolite repression, only the expression of the gene is inhibited irrespective of the cell growth. As stated above, the Escherichia coli used in Japanese Patent Publication No. 42555/1986 is controlled by glucose both in the amount of the cells recovered and the enzyme activity.
It is considered that the phenomenon disclosed in Patent Publication No. 42555/1986 is entirely different from "catabolite repression" and is quite unique and specific only for Escherichia coli strains cited in the above patent document.
As far as the present inventors know, there has been no disclosure on the cultivation methods of recombinant Escherichia coli strains utilizing the tna mediated gene promoter expression system.